We propose to continue studies on the mechanisms of eukaryotic gene transcription initiation and mRNA splicing using globin genes as a model system. The approach is to compare the expression of cloned globin genes isolated from patients with inherited disorders in globin gene expression (thalassemia), or genes which have been mutagenized in vitro, with the expression of normal globin genes. In order to obtain single base mutations in promoter sequences, we propose to develop a new method for directed mutagenesis which involves in vitro chemical mutagenesis and the isolation of the mutant DNA fragments by denaturing gradient polyacrylamide gel electrophoresis. Defects in transcription will be analyzed by introducing the altered genes into mammalian cells in culture using an SV40-derived expression vector, and by studying transcription of the altered genes in vitro. Splicing defects will be analyzed by using mammalian cell transfection procedures, by injection of in vitro synthesized globin pre-mRNAs into Xenopus oocytes, or by addition of the synthetic pre-mRNA to cell-free splicing extracts. The objectives of this research are to identify the DNA and RNA sequences required for transcription and splicing. Interactions between cis and trans-acting viral transcription control elements and globin gene promoters will be studied in vivo and in vitro to determine the mechanism by which these control elements activate transcription from both viral and cellular genes. This will be accomplished by cotransfecting cloned viral and globin genes into mammalian cells in culture and then analyzing globin gene transcription. In order to locate the sites of action of the viral control elements within the globin gene promoters, both naturally occurring and in vitro mutagenized promoter mutants will be examined in the cotransfection assay. It is possible that these sites also interact with cellular factors that activate transcription of globin genes in erythroid cells. The long term objective of this research is to gain a better understanding of the interactions between globin gene promoters and factors which mediate transcription.